Search

CD80 (B7-1) and CD86 (B7-2) on APC provide a major costimulatory signal through interactions with CD28 on T cells. Absent from resting human T cells, CD86 is up-regulated early upon T cell activation, whereas CD80 expression appears later. Whereas T cell expression of CD80 has been implicated in costimulation, the functional significance of CD86 expression on T cells is unclear. We now demonstrate that CD86 expressed on human CD4+ T cell clones does not provide a costimulatory signal for other CD4+ T cell clones. Binding studies using CD28-Ig and CTLA-4-Ig fusion proteins demonstrate that CD86 expressed on T cells has significantly reduced binding affinity for CTLA-4 and no detectable binding to CD28. Biochemical analysis demonstrates that post-translational modifications of CD86 in human T cells are different from those of CD86-transfected Chinese hamster ovary cells or EBV-transformed B cells, in that T cells express a hypoglycosylated form of CD86 on the surface membrane. Thus, our results suggest that while CD86 is expressed on a number of different cell types, its costimulatory function and affinity for its ligands may be regulated by cell type-specific post-translational modifications.

Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease inducible in susceptible animals by myelin Ag-specific CD4+ Th1 cells. The mechanisms by which these cells induce inflammation and demyelination in the central nervous system (CNS) are incompletely understood. To determine the roles of Fas and FasL in the involvement of CNS autoimmune injury, we determined susceptibility to EAE of Fas-or FasL-deficient mice. Compared with wild-type mice, mice expressing lpr (Fas) and gld (FasL) mutations were relatively resistant to the development of clinical EAE, and this correlated with fewer inflammatory infiltrates and cells undergoing apoptosis in the CNS of the mutant mice. The gld and lpr mice, however, developed significant T cell responses with production of Th1 cytokines in response to the encephalitogenic myelin peptide. These results suggest that the Fas/FasL pathway plays a critical role in the development of EAE probably by mediating apoptosis within the target tissue.

The B7 family of costimulatory molecules provides the second signal necessary for activation of T cells. In the absence of the second signal, responding T cells become anergic. Although predominantly expressed on professional APCs, recent evidence shows that the B7 molecules are also expressed on T cells. To study the functions of B7 molecules on T cells, we transfected murine B7.1 (CD80) and B7.2 (CD86) cDNAs into the EL4 T cell thymoma cell line and examined the transfectants for their ability to costimulate T cell proliferation in vitro and to induce antitumor immunity in vivo. Here we show that although EL4-B7.1 cells costimulate T cells and induce tumor regression, EL4-B7.2 transfectants failed to costimulate T cell proliferation or induce tumor regression. To understand the cellular basis for this difference, we examined the binding of EL4-B7.1 and EL4-B7.2 to CTLA4 and CD28. Whereas EL4-B7.1 cells bound both CTLA4-Ig and CD28-Ig, EL4-B7.2 transfectants preferentially bound CTLA4-Ig, but not CD28-Ig. Similar binding data were obtained with freshly isolated murine T cells, which have been shown to constitutively express B7.2. Our data suggest, therefore, that B7.2 expressed on T cells may not costimulate but instead inhibit the T cell response by preferential binding to CTLA4.

Experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis, is a T cell-mediated autoimmune disease that can be induced in experimental animals by immunization with myelin Ags. Inbred strains of mice show varying degrees of susceptibility to EAE, indicating that susceptibility is an inherited trait. To define the genetic factors that control susceptibility to EAE, we performed linkage analysis on the first backcross (BC1) between highly susceptible SJL/J mice and resistant B10.S mice, both of which are of the H-2s haplotype. Mice were immunized for disease with encephalitogenic myelin proteolipid protein peptide 139 to 151, and analysis was performed on 68 backcross mice showing the severe disease phenotype (disease score > or = 3)and 68 backcross mice of the resistant phenotype (no clinical or histologic signs of disease) using microsatellite markers covering >98% of the genome. We found the strongest linkage (p = 0.001) with clinical disease at two loci: one at the telomeric end of chromosome 2, and another near the center of chromosome 3. In addition, several other regions showing some evidence of linkage (p < or = 0.05) with clinical disease were found.

To understand and develop strategies to intervene in autoimmune responses to myelin proteolipid protein (PLP), encephalitogenic epitopes must be identified. To expedite the identification of potentially immunogenic and encephalitogenic epitopes of PLP, overlapping synthetic 20-mer PLP peptides covering the whole PLP molecule were screened for their ability to bind to purified mouse I-Ad, I-Ak, and I-As molecules. The peptides that bound to the I-A molecules were tested for their ability to induce immune responses in corresponding inbred mouse strains. Immunogenic peptides were then tested for their ability to induce experimental autoimmune encephalomyelitis. Moderate to strong I-A binding was essential for development of immune responses, but immunogenicity was not sufficient for encephalitogenicity. Rather, encephalitogenic epitopes clustered in three regions of the molecule, namely within residues 40-70, 100-119, and 178-209. These were also the regions of the PLP that showed the greatest promiscuity in binding to I-A molecules. Except for PLP 139-151, which is an encephalitogenic determinant in mice expressing I-As, all encephalitogenic epitopes of PLP previously identified, regardless of their MHC class II restriction, are located within or adjacent to these epitope clusters. None of the encephalitogenic epitopes occur in regions of the molecule that have a high degree of homology with the neuronal M6a protein, a member of the DM20/PLP superfamily. Atypical clinical and histologic patterns of experimental autoimmune encephalomyelitis were observed in some strains of mice sensitized with certain PLP peptides and may reflect induction of T cells with different disease-inducing potentials.

We investigated the immune response to proteolipid protein (PLP), the most abundant central nervous system myelin protein in humans. A total of 8207 short-term T cell lines were generated from 49 individuals, 39 patients with multiple sclerosis and 10 control subjects. As we have reported previously, the frequency of PLP-reactive T cells did not differ between the two groups. To determine immunodominant PLP epitopes, proliferative responses of 971 PLP-specific lines were tested with 27 overlapping 20-amino acid peptides encompassing the human PLP sequence and the binding affinities of the PLP peptides to DRB5*0101 and DRB1*1501, DR2 MHC class II isotypes associated with multiple sclerosis, were determined. The T cell response after primary PLP stimulation was focused on two immunodominant epitopes comprising residues p30-49 and p180-199. These two fragments were recognized after processing of native protein by APCs and were situated in hydrophilic regions of PLP exhibiting only moderate affinity to DR2 molecules. In contrast, when T cells from DR2+ subjects were stimulated initially by individual synthetic peptides with either high or low affinity to DRB5*0101 and DRB1*1501 isotypes, additional cryptic epitopes were recognized. MHC restriction of lines specific for the cryptic PLP epitopes were related to binding affinity to DR2 isotypes. Our results indicate that protein Ags are recognized in vivo as immunodominant epitopes after Ag processing by APCs and as cryptic epitopes after processing, presumably by extracellular proteolytic enzymes.

Conditioned medium from Ag-specific suppressor T cell hybridomas contains soluble factors (TsF) that modulate immune responses in an Ag-specific manner. We previously generated a series of TCR-alpha- and TCR-beta- expression variants from a 4-hydroxy-3-nitrophenyl acetyl (NP)-specific inducer suppressor T cell hybridoma and demonstrated that loss of TCR alpha-chain mRNA, but not TCR-beta chain mRNA, was accompanied by concomitant loss of suppressor bioactivity. Suppressor factor bioactivity was restored by expression of TCR alpha-chain cDNA, suggesting that the TCR alpha-chain plays a critical role in Ag-specific suppressor cell function. We have now transfected TCR alpha-chain from a Th cell clone specific for arsanylated peptides plus I-Ad into a TCR-alpha- derivative of an NP-specific inducer suppressor T cell hybridoma. The transfectants expressed a new hybrid TCR-alpha beta complex and produced soluble factors that suppressed azobenzenearsonate hapten (ABA) but not NP delayed-type hypersensitivity responses. These supernatants mediated suppression of the induction, but not the effector phase of the delayed-type hypersensitivity reaction. In reciprocal experiments we transfected a TCR alpha-chain from an NP-specific suppressor T cell hybridoma into a TCR-alpha- hybridoma derived from the ABA-specific Th cell hybridoma. The NP-specific TCR alpha-chain was expressed in the Th cell hybridoma, but the supernatant from this transfectant did not suppress DTH responses to either NP or ABA. However, the latter supernatants, when combined with cell lysates derived from a TCR-alpha- Ts hybridoma, specifically suppress NP DTH responses. These data are consistent with the interpretation that TCR alpha-chain imparts Ag specificity to the suppressor molecule and a second, yet undefined, component produced by the Ts hybridoma controls the immunoregulatory bioactivity.

Previous studies utilizing NP (4-hydroxy, 3-nitrophenyl acetyl hapten)-specific, T suppressor hybridomas have indicated that expression of TCR-alpha, but not TCR-beta, mRNA is required for expression of Ag-specific suppressor factor bioactivity. Suppressor-effector factor has been shown to be Ag specific and I-J restricted. Although the expression of TCR-alpha mRNA was necessary for suppressor activity, the role of TCR-alpha, as it pertained to the functional properties of T cell suppressor factor (TsF), was not established. To determine which properties of TsF could be accounted for by TCR-alpha expression, TCR-alpha cDNA, derived from NP-specific, suppressor T cell (Ts) hybridomas, was transfected into recipient Ts hybridomas of a second Ag specificity. The resulting heterologous transfectants displayed NP-specific, genetically restricted TsF activity. The Ag specificity corresponded to that of the TCR-alpha donor; however, the genetic restriction was influenced by the recipient cell, implying that TCR-alpha did not control genetic restriction of the TsF. Examination of TCR-beta expression in one of the MHC-restricted transfectants indicated that the genetic restriction of TsF could not be accounted for by TCR-beta gene products. The data support the conclusion that TCR-alpha expression is not only obligate for TsF bioactivity, but that the Ag specificity of the TCR-alpha dictates the Ag specificity of the resulting suppressor factor.

Previously, six T cell clones, which are specific for an encephalitogenic determinant of myelin proteolipid protein (PLP) peptide residues 139 to 151 (HSLGKWLGHPDKF), were derived from SJL mice and shown to use diverse TCR genes. To design TCR antagonist peptides that could interfere with the activation of these clones in vitro and inhibit experimental allergic encephalomyelitis (EAE) in vivo, we first determined the TCR and MHC contact residues of the encephalitogenic peptide. The analysis indicated that residues 144 (tryptophan) and 147 (histidine) were the TCR binding sites and that residues 145 (leucine) and 148 (proline) were important for MHC class II (IAs) binding. On the basis of this information, a peptide analogue (leucine 144/arginine 147), in which both of the major TCR contact residues were substituted, was synthesized. This analogue acts as a TCR antagonist for the panel of PLP 139-151-specific T cell clones, does not cause EAE by itself, blocks the induction of disease by the native 139-151 peptide, and prevents clinical disease progression if administered at the first signs of disease. Thus, although multiple TCR genes are used by PLP 139-151-specific clones, a single peptide analogue can interfere with the disease process. This approach should be feasible for designing peptide analogues that can be tested for therapeutic efficacy in human autoimmune diseases in which the pathogenic Ags are known and TCR use is diverse.

We have derived a panel of CD4+, TCR-alpha/beta + T cell clones from SJL (H-2s) mice specific for an encephalitogenic determinant of myelin proteolipid protein (PLP) 139-151 (HSLGKWLGHPDKF). All the clones are Ag specific and IAs restricted, but they show heterogeneity in their ability to induce experimental allergic encephalomyelitis (EAE), i.e., one group induces EAE in naive mice, a second group induces disease only in mice that are pretreated with pertussis and irradiation, whereas a third group is essentially nonencephalitogenic. To determine the basis for this functional heterogeneity, the clones were tested for the expression of adhesion molecules and cytokines and for Ag-specific cytolytic activity. All of the clones expressed comparable levels of LFA-1 and CD44 but lacked expression of Mel 14. However, those clones that induced EAE only in irradiation- and pertussis-treated recipients did not express VLA4. Because pretreatment with pertussis has been suggested to increase permeability of the blood-brain barrier and facilitate migration of T cells into the central nervous system, the absence of VLA4 on this group of clones may account for the need for pretreatment to induce EAE. The nonencephalitogenic clones expressed all of the adhesion molecules tested but were not cytolytic in vitro and failed to produce one or more of the proinflammatory cytokines after Ag-specific stimulation. One nonencephalitogenic clone that did not produce many cytokines on activation with specific Ag, however, could be activated with Con A to express mRNA for most cytokines and this was accompanied by a concomitant change in the encephalitogenic potency of this clone. These results suggest that adhesion molecules and cytokines both play a critical role in the encephalitogenicity of PLP peptide-specific T cell clones. Furthermore, the nonencephalitogenicity of some clones may be related to a defect in Ag-mediated activation.

To determine whether there is predominance of T cells expressing a particular TCR V beta chain in the inflammatory lesions of an autoimmune disease model, TCR expression was analyzed in central nervous system (CNS) tissues of mice with experimental allergic encephalomyelitis (EAE). Acute EAE was induced in SJL/J mice either by sensitization with a synthetic peptide corresponding to myelin proteolipid protein residues 139-151 or by adoptive transfer of myelin proteolipid protein peptide 139-151-specific encephalitogenic T cell clones. Mice were killed when they showed clinical signs of EAE or by 40 days after sensitization or T cell transfer. Cryostat CNS and lymphoid tissue sections were immunostained with a panel of mAb to T cell markers and proportions of stained cells were counted in inflammatory foci. In mice with both actively induced and adoptively transferred EAE, infiltrates consisted of many CD3+, TCR alpha beta+, and CD4+ cells, fewer CD8+ cells, and small numbers of TCR gamma delta+ cells. Approximately 30% of CD45+ leukocytes in the inflammatory foci were T cells. Cells expressing TCR V beta 2, 3, 4, 6, 7 and 14 were detected in the infiltrates, whereas TCR V beta 8 and 11, which that are deleted in SJL mice, were absent. When EAE was induced by transfer of T cell clones that use either V beta 2, 6, 10, or 17, there was also a heterogeneous accumulation of T cells in the lesions. Similar proportions of TCR V beta+ and gamma delta+ cells were detected in EAE lesions and in the spleens of the mice. Thus, at the time that clinical signs are present in acute EAE, peripherally derived, heterogeneous TCR V beta+ cells are found in CNS lesions, even when the immune response is initiated to a short peptide Ag or by a T cell clone using a single TCR V beta.

We previously described a synthetic peptide of myelin proteolipid protein (PLP), peptide 139-151, which induces experimental allergic encephalomyelitis in SJL/J (H-2s) mice. We have now identified an additional determinant, PLP residues 178-191, that is also a potent encephalitogen in this strain. When PLP peptide 178-191 was compared with peptide 139-151 on an equimolar basis, the day of onset of disease induced by PLP 178-191 was earlier, but the incidence, severity, and histologic features were indistinguishable. Lymph node cells from animals immunized with the whole PLP molecule responded to both PLP 178-191 and 139-151, suggesting immunologic codominance of the two epitopes. PLP 178-191 elicited stronger proliferative responses and this may relate to the earlier onset of disease induced with this peptide. Two CD4+, peptide-specific, I-A(s)-restricted T cell lines, selected by stimulation of lymph node cells with either PLP 178-191 or 139-151, were each encephalitogenic in naive syngeneic mice. The presence of multiple encephalitogenic codominant PLP epitopes within a single mouse strain emphasizes the complexity of the immune response to PLP and its potential as a target Ag in autoimmune demyelinating diseases.

Proteolipid protein (PLP) is the major protein of central nervous system myelin. SJL (H-2s) mice immunized with a synthetic peptide corresponding to PLP residues 139-151 develop acute EAE. In this study, 6 IAs-restricted, CD4+, TCR alpha beta-bearing T cell clones were derived from SJL/J mice after immunization with this synthetic peptide. The clones responded in in vitro proliferative assays to the whole PLP molecule and to PLP peptide 139-151, but not to irrelevant Ag. They also responded to truncated and overlapping forms of the peptide but five distinct reactivity patterns were observed using these peptides. A panel of anti-TCR V beta mAb and TCR V beta-specific cDNA probes were used to determine the TCR V beta usage of the clones. Five clones were found to use four different V beta (V beta 2, V beta 6, V beta 10, or V beta 17a), whereas the V beta on the sixth clone could not be identified. Five of the clones induced EAE of varying severity upon adoptive transfer into naive syngeneic mice or mice pretreated with irradiation and pertussis and one clone was nonencephalitogenic. The Ag-specific proliferative response of all but the nonencephalitogenic clone could be blocked by an anti-CD4 mAb. Thus, the clones showed differences in their fine specifity, TCR V beta usage, sensitivity to antibody blocking, and encephalitogenic potency. These data demonstrate that the T cell response to the encephalitogenic PLP peptide 139-151 is heterogeneous.

A T cell hybridoma producing a T suppressor factor (TsF) with specificity for the hapten nitrophenyl was converted to long term growth in serum-free medium and its product tested by serology, bioactivity, and Western blot analysis. Results indicated that Ag-specific suppressive activity was present in serum-free medium and this TsF could exhibit the characteristics ascribed to it by various groups: it could bind nominal Ag with specificity, it was bound by anti-TsF mAb, and it could mediate Ag-specific suppression both in vivo and in vitro. Western blot and SDS-PAGE analysis of this purified TsF revealed a 43-kDa single chain protein.

We have examined the expression of TCR genes in 4-hydroxy-3-nitrophenyl-acetyl (NP)-specific Ts cell hybridomas. Each of three independently isolated hybridomas expressed in-frame TCR alpha-chain rearrangements derived from the original suppressor Ts cell. Different V alpha and J alpha gene segments were rearranged and expressed in each Ts cell line. The only TCR beta-chain expressed in these cells was derived from the BW5147 fusion partner. Expression of the BW5147 beta-chain was found to correlate with cell surface Ag binding, inasmuch as subclones derived from one of the original Ts lines expressed greatly reduced levels of beta-chain mRNA and no longer bound to NP-coupled RBC. Subclones that continued to express beta-chain mRNA did bind to NP-coupled RBC. This suggests that the Ag receptor on Ts hybridomas is a TCR-alpha beta dimer composed of a unique alpha-chain and the BW5147 beta-chain. Ag binding could be modulated by preincubation of Ts hybridoma cells with anti-TCR-alpha beta antibody, thereby supporting this conclusion. Suppressor factor activity was measured in the conditioned media of Ts subclones that differed by 250-fold in levels of beta-chain mRNA expression. No difference in suppressor factor activity was found; conditioned media from these subclones suppressed both plaque-forming cell responses and delayed-type hypersensitivity responses at approximately equivalent dilutions. Suppressor factor activity in the conditioned media of both a beta-chain negative subclone and a beta-chain positive subclone could be absorbed with an antibody that recognizes the TCR alpha-chain, but not with an antibody that recognizes the TCR beta-chain. We conclude that suppressor factor activity in the conditioned media of these Ts hybridomas is not derived from surface TCR-alpha beta receptors, although it does share TCR alpha-chain determinants.

TCA3 is a cDNA originally isolated from activated T cells. Transcription of this gene has been shown to correlate with Ag-induced cellular activation of both T cells and mast cells. Based on the predicted amino acid sequence encoded by the cDNA, we previously proposed that TCA3 represents a cytokine. In this report we have used rDNA technology to express TCA3 in two mammalian cell lines. In both cases, TCA3 was expressed as a secreted molecule with an apparent molecular mass of 16 kDa. Digestion of the (rTCA3) with the enzyme N-glycanase revealed that approximately 8 kDa is caused by N-linked glycosylation. Intradermal injection of rTCA3 into mouse footpads resulted in a rapid swelling response. The sites of injection were characterized histologically by a local accumulation of neutrophils. These findings are discussed with particular attention to a family of related proteins, some of whose members also have inflammatory properties.

Eight different Ts cell hybridomas (including inducer (Ts1) and effector (Ts3) suppressor cells) specific for the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten were tested for their ability to respond to Ag or anti-CD3 antibody in a growth-inhibition assay. Results suggest that the expression of the TCR-CD3 complex on Ts hybridomas is required for the Ag or anti-CD3-mediated growth inhibition. One of the CD3+, Ts hybridomas (CKB-Ts3-9.H3) was tested in detail; this CD4- effector suppressor cell hybridoma showed specific inhibition of growth in the presence of NP or NIP-coupled protein conjugates but not in the presence of other irrelevant hapten-protein conjugates. In addition, growth of this hybridoma was specifically inhibited by anti-CD3 and anti-TCR-alpha/beta antibodies but not by control hamster antibodies. In order to study the role of MHC molecules in Ag-mediated growth inhibition, Ts cell hybridomas were incubated with Ag (NP-keyhole limpet hemocyanin) in the presence of spleen cells from various H-2 congenic strains. The results suggest that the Ts hybridomas that express donor Ts-derived TCR beta-chain recognize Ag in an MHC-restricted manner, whereas the two Ts3 hybridomas that utilize BW5147-derived TCR-beta recognize Ag in H-2 unrestricted way. Co-incubation of anti-CD3 and anti-TCR-alpha/beta antibodies with specific Ag enhanced the Ag-mediated growth inhibition, whereas anti-LFA-1 antibody completely blocked the Ag-mediated effect. The combined data suggest that, like Th hybridomas, expression of CD3-associated-TCR complex is essential for the Ag responsiveness of Ts cell hybridomas.

Ts1, or inducer suppressor T cells, share many phenotypic and functional characteristics with helper/inducer subset of T cells. In order to evaluate the relationship between these cell types, we made a series of new Ts1 hybridomas by the fusion of Ts1 cells with the functionally TCR alpha/beta-negative BW thymoma (BW 1100). Three Ts1 hybridomas (CKB-Ts1-38, CKB-Ts1-53, and CKB-Ts1-81) were established that express TCR and produce Ag-specific suppressor factors constitutively, thus making it possible to study the nature and specificity of Ag receptors, MHC restriction, and lymphokine production by the Ts1 hybridomas. Results presented in this report demonstrate that all the Ts1 hybridomas described here express CD3-associated TCR-alpha beta. These three Ts1 hybridomas recognize Ag (NP-KLH) specifically in a growth inhibition assay and this recognition is restricted by IE molecules. Two of the hybridomas also produce IL-2 or IL-2 and IL-4 upon Ag-specific activation. Thus, by these three criteria the Ts1 hybridomas appear indistinguishable from Th cells. These three Ts1 hybridomas, however, release suppressor factors (TsF1) in the supernatant that suppress both in vivo DTH and in vitro PFC responses in an Ag-specific manner. Like the TsF1 factors characterized previously, the suppression mediated by these factors are Igh restricted and lack H-2 restriction. These factors mediate suppression when given in the induction phase but not during the effector phase of the immune response. The TsF1 factors are absorbed by Ag (NP-BSA), and anti-TCR affinity columns and the suppressor activity can be recovered by elution. The data are consistent with the interpretation that Ts1 inducer-suppressor T cells are related to Th cells; the feature that distinguishes these cells is the ability to produce Ag-binding factors that specifically suppress immune responses.

Natural Killer (NK) T cells are a specialized T cell population that co-expresses receptors of the NK lineage with the alpha / beta TCR receptor and other T cell surface markers. Their functions, regulation and relationship to other cells in the immune system are not fully understood. This report demonstrates that tumor-bearing C57BL / 6 mice have a population of NKT cells that co-express CD8 and CD161 (NK1.1) surface markers. These cells are maintained in long-term culture with T helper 2 (Th2) cytokine interleukin-4 (IL-4), but produce large amounts of Th1 cytokine interferon-gamma (IFN-gamma) following activation. NK1.1(+)CD8(+) T cells show a potent NK-like cytotoxic activity against multiple tumor targets, and lysis is independent of major histocompatibility complex (MHC)-class I or non-classical MHC-class I molecules (Qa, TL). The NK1.1(+)CD8(+) T cells express Vbeta14 chain of the TCR. These NKT cells are not CD1d restricted, and their cytotoxic activity is CD1d independent. Therefore, they represent a unique subset of T cells with an unknown restriction element which produce large quantities of IFN-gamma following expansion with IL-4. Furthermore, their cytotoxic activity is enhanced by B7 co-stimulatory molecules present on tumor cells. CD161(+) T cells that are expanded in tumor-bearing hosts may function as a part of the innate immune system with potential role(s) in tumor surveillance.

A clear understanding of the events surrounding the selection of autoreactive T cells in the thymus and their regulation in the periphery has eluded immunologists for years. However, recent work examining the expression of tissue-specific antigens in the thymus and the biochemistry of disease associated MHC alleles has provided important clues into the generation of the autoreactive T cell repertoire in the thymus. In addition, recent studies focusing on the role of immunoregulatory cytokines and cross-reactive peptide ligands has provided information regarding both the regulation and activation of autoreactive cells in the periphery. An improved understanding of the selection and regulation of autoreactive T cells will undoubtedly aid in the development of strategies for treating autoimmune disease.

The autoreactive T cells that escape central tolerance and form the peripheral self-reactive repertoire determine both susceptibility to autoimmune disease and the epitope dominance of a specific autoantigen. SJL (H-2(s)) mice are highly susceptible to the induction of experimental autoimmune encephalomyelitis (EAE) with myelin proteolipid protein (PLP). The two major encephalitogenic epitopes of PLP (PLP 139-151 and PLP 178-191) bind to IA(s) with similar affinity; however, the immune response to the PLP 139-151 epitope is always dominant. The immunodominance of the PLP 139-151 epitope in SJL mice appears to be due to the presence of expanded numbers of T cells (frequency of 1/20,000 CD4(+) cells) reactive to PLP 139-151 in the peripheral repertoire of naive mice. Neither the PLP autoantigen nor infectious environmental agents appear to be responsible for this expanded repertoire, as endogenous PLP 139-151 reactivity is found in both PLP-deficient and germ-free mice. The high frequency of PLP 139-151-reactive T cells in SJL mice is partly due to lack of thymic deletion to PLP 139-151, as the DM20 isoform of PLP (which lacks residues 116-150) is more abundantly expressed in the thymus than full-length PLP. Reexpression of PLP 139-151 in the embryonic thymus results in a significant reduction of PLP 139-151-reactive precursors in naive mice. Thus, escape from central tolerance, combined with peripheral expansion by cross-reactive antigen(s), appears to be responsible for the high frequency of PLP 139-151-reactive T cells.

Oral administration of Ag leads to systemic unresponsiveness (oral tolerance) to the fed Ag. Oral tolerance is mediated through active suppression by Th2 or TGF-beta-secreting cells or clonal anergy/deletion, depending on the Ag dose used, with low dose favoring active suppression and high dose favoring anergy/deletion. The nature of APC and inductive events leading to the generation of oral tolerance have not been well defined. To determine the role of costimulatory molecules in the induction of oral tolerance, we have tested the effect of anti-B7.1 or anti-B7.2 mAb on the induction of tolerance by both high and low dose Ag feeding regimens. Our results show that the B7.2 molecule is critical for the induction of low-dose oral tolerance. Injection of anti-B7.2 but not anti-B7.1 intact Ab or Fab fragments inhibited the oral tolerance induced by low-dose (0.5 mg) but not high-dose OVA (25 mg) feeding. In addition, anti-B7.2, but not anti-B7.1, inhibited secretion of TGF-beta, one of the primary cytokines that mediates low-dose oral tolerance. Finally, in the in vivo model of experimental allergic encephalomyelitis, anti-B7.2 mAb treatment abrogated protection offered against disease by low-dose myelin basic protein feeding, while anti-B7.1 had no effect. Anti B7.2 had no effect on disease suppression by high-dose oral Ag. These data demonstrate that B7.2 costimulatory molecules play an essential role in the induction of low-dose oral tolerance.

Cross-reactivity with environmental antigens has been postulated as a mechanism responsible for the induction of autoimmune disease. Experimental autoimmune encephalomyelitis is a T cell-mediated autoimmune disease model inducible in susceptible strains of laboratory animals by immunization with protein constituents of myelin. We used myelin proteolipid protein (PLP) peptide 139-151 and its analogues to define motifs to search a protein database for structural homologues of PLP139-151 and identified five peptides derived from microbial Ags that elicit immune responses that cross-react with this self peptide. Exposure of naive SJL mice to the cross-reactive environmental peptides alone was insufficient to induce autoimmune disease even when animals were treated with Ag-nonspecific stimuli (superantigen or LPS). However, immunization of SJL mice with suboptimal doses of PLP139-151 after priming with cross-reactive environmental peptides consistently induced experimental autoimmune encephalomyelitis. Furthermore, T cell lines from mice immunized with cross-reactive environmental peptides and restimulated in vitro with PLP139-151 could induce disease upon transfer into naive recipients. These data suggest that expansion by self Ag is required to break the threshold to autoimmune disease in animals primed with cross-reactive peptides.

Experimental autoimmune encephalomyelitis (EAE) and other organ-specific autoimmune diseases are induced by autoantigen-specific Th1 cells. In contrast, transfer of autoantigen-reactive Th2 cells that produce IL-4 and IL-10 can prevent and/or reverse EAE. The relative roles of these two Th2 cytokines in the regulation of EAE has not been evaluated. Utilizing IL-4 and IL-10 knockout mice deficient for these cytokines and IL-10 and IL-4 transgenic mice overexpressing these cytokines, we demonstrate that IL-10-deficient mice (IL-10(-/-)) are more susceptible and develop a more severe EAE when compared with IL-4-deficient mice (IL-4(-/-)) or wild-type mice. T cells from IL-10(-/-) mice exhibit a stronger Ag-specific proliferation, produce more proinflammatory cytokines (IFN-gamma and TNF-alpha) when stimulated with an encephalitogenic peptide, and induce very severe EAE upon transfer into wild-type mice. In contrast, while IL-4 transgenic mice develop similar disease compared with their nontransgenic littermates, mice transgenic for IL-10 are completely resistant to the development of EAE. Taken together, our data suggest that IL-10 plays a more critical role in the regulation of EAE by regulating autopathogenic Th1 responses.

T lymphocytes bearing alpha/beta TCR recognize antigens in the context of self MHC molecules, and this recognition leads to growth, differentiation, and effector functions. Recently, it has become clear that altered peptides generated by single amino acid substitution of the antigenic peptide can alter the patterns of differentiation and effector functions of the responding T lymphocytes. By defining the pattern of recognition and residues of the cognate ligand that bind to the TCR, altered peptide ligands (APLs) have been generated by selectively substituting the TCR contact residues in the antigenic peptide. These APLs have been utilized in vitro to study the biology of T cell function and alterations in the T cell signaling pathway. In vivo APLs have been utilized to study the mechanism of positive selection in the thymus and in regulation of autoimmune diseases. With this basic knowledge, APLs that can either hypo- or hyper-stimulate T cell function can be generated that can specifically alter (inhibit or enhance) immune responses in vivo in autoimmune diseases and cancers.

Experimental autoimmune encephalomyelitis is a disease of the central nervous system that can be readily induced in a variety of species by immunization with myelin proteins. It is one of the most commonly studied models of cell-mediated autoimmune disease and consequently has been important in the elucidation of many immunological functions in vivo. Nevertheless, very little is understood about the genetic and environmental factors that control the disease. Several groups have begun undertaking systematic genetic analyses in mice to identify loci that associate with increased susceptibility to disease. In this review, we will summarize the work that has been done to understand the genetic predisposition that makes certain inbred animals susceptible to disease and others resistant, and we will discuss some of the difficulties in the genetic analysis that have arisen due to the complexities of this disease model.

A monoclonal antibody (mAb), B16G, was raised from BALB/c mice immunized with affinity-purified T suppressor factors (TsF) specific for the murine mastocytoma P815. This mAb was found to bind to polyclonal TsF isolated from the spleens of tumor-bearing animals, and to the TsF released from a P815-specific T cell hybridoma. In this study, B16G was tested for its reactivity with TsF produced in the 4-hydroxy-3-nitrophenyl acetyl hapten system. The factors from three types of suppressor T cell hybridomas, each representing the immortalized analogues of the inducer T suppressor cell (Ts1), transducer suppressor cell (Ts2), and effector suppressor cell (Ts3) network populations, were tested. B16G was found to be reactive with two sources of TsF1 as assayed by enzyme-linked immunosorbent assay and delayed-type hypersensitivity bioassay. By contrast, TsF2 and TsF3 were nonreactive with B16G. These results indicate that B16G recognizes class-specific suppressor factor determinants, and that the transducer/effector factors of the network are apparently serologically distinct. Because the B16G mAb fails to recognize 4-hydroxy-3-nitro-phenyl acetyl-specific TsF3 that share idiotype-related determinants with TsF1 yet binds to TsF1 molecules that have interacted with antigen, the binding is apparently independent of the site of antigen recognition. Additionally, the results show that the tumor-specific TsF1 raised in one suppressor system share serologic determinants with anti-hapten TsF1 raised in another.

Five different Ag-binding suppressor factors from two types of hapten-specific Ts cell hybridomas (TsF1 inducer and TsF3 effector factors) were bound by an anti-lipomodulin mAb (141-B9), that crossreacts with rodent glycosylation inhibition factor (GIF). The Ag-specific suppressor activity in these hybridoma supernatants was bound by anti-lipomodulin columns and could be recovered by elution at acid pH. Additional evidence for the expression of lipomodulin/GIF activity on these TsF molecules was demonstrated by the ability of the eluted fractions to inhibit the glycosylation of IgE-binding peptides during their biosynthesis. The same biologic activity is associated with GIF and lipomodulin. The relationship between TsF and lipomodulin/GIF was confirmed in a serologic assay, which showed that TsF1 and TsF3 molecules, whether purified over Ag, anti-IJ or anti-TsF columns, are recognized by the mAb. 141-B9. The combined results indicate that Ag-binding Ts factors share a common antigenic determinant with phospholipase inhibitory proteins such as lipomodulin and GIF. In addition, the demonstration of glycosylation regulatory activity carried on these TsF molecules suggests a possible mode for their bioactivity.

We previously screened a series of macrophage hybridomas derived from fusion of P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells for their ability to induce I-J restricted Ts cell responses. One Ia+ macrophage clone (63) consistently induced Ag-specific, I-J-restricted Ts. To evaluate whether macrophage hybridoma 63 also induced delayed-type hypersensitivity (DTH) immunity, mice were immunized with hapten-coupled macrophage hybridoma cells. Hapten-coupled splenic adherent cells and control macrophage hybridomas induced significant primary DTH responses, whereas hapten-coupled macrophage 63 induced little or no immunity when injected into H-2 compatible hosts. However, macrophage hybridoma 63 specifically activated I-Ak, I-Ad, or I-Ed restricted T cell hybridomas/clones, in vitro in the presence of appropriate Ag. Three different strategies designed to eliminate suppressor cell activity were successfully used to demonstrate that hapten-coupled macrophage 63 could also induce in vivo immunity. First, after immunization with hapten-coupled macrophages, mice were treated with cyclophosphamide. Second, macrophage 63 was treated with anti-IJ idiotype antibody before 4-hydroxy-3-nitrophenyl acetyl hapten (NP) coupling. Finally, haptenated macrophages were injected into I-A compatible but I-J incompatible recipients. These protocols are known to inhibit the induction of Ts activity, thus these results indirectly suggest that there is stimultaneous generation of Ts activity in vivo. The latter hypothesis was tested in adoptive transfer experiments. Transfer of lymph node cells from NP-63 primed B10.BR (H-2k) mice induced immunity in naive 4R animals, whereas the same number of immune cells suppressed NP-induced DTH responses in 5R mice. The combined results indicate that a cloned macrophage line can activate both Th and Ts cells. Macrophages which induce Ts activity may be responsible for maintaining the balance of immunity vs suppression. The data support the hypothesis that IJ interacting molecules (IJ-IM) expressed on macrophages are critical for induction of suppressor cell activity.

In the 4-hydroxy-3-nitrophenyl acetyl (NP) suppressor system, third order suppressor cells (Ts3) subset of suppressor cells is generated after Ag priming, but, in order to express suppressor activity, these cells need to be further activated or triggered with a specific second order suppressor factor. By in vitro activation of Ts3-containing lymph node cells or a pTs3 hybridoma we now show that macrophages are also required for Ts3 activation. In addition, we demonstrate that IJ genetic restrictions control this activation process. Furthermore, we directly demonstrate Ts3 activation using cloned macrophage hybridoma cells. To further investigate the interactions between Ts3 cells and the accessory cells involved in their activation, we attempted to block the second order suppressor factor mediated activation of Ts3 cells with antibodies. The activation of Ts3 cells can be blocked by the addition of anti-IJ, anti-IJ idiotype or anti-NPb idiotype antibodies, but not by anti-CD8, anti-IA, or anti-IE antibodies. Anti-IJ mAb blocked Ts3 activation at the lymphocyte level whereas anti-IJ idiotype blocked activation at the accessory cell level. Finally we tested, whether these antibodies can also directly activate primed Ts3 cells. We demonstrate that cross-linked anti-IJ, anti-NPb and anti-CD3 antibodies can activate Ts3 cells. The results are discussed in terms of receptor-ligand structures on Ts and accessory cells which are required for the activation of Ts3 cells.

Blood leukocytes from cattle with bovine ocular squamous cell carcinoma (BOSCC) react with extracts of BOSCC, and blood leukocytes from cattle with cutaneous papillomas react with extracts of papillomas in the erythrocyte rosette (E-rosette) augmentation test. Sera from cattle with BOSCC, with cutaneous papillomas, and with ocular papillomas and sera from lesion-free cattle were tested for their ability to block antigen-induced E-rosette augmentation and E-rosette augmentation induced by phytohemagglutinin. Autologous serum from 16 BOSCC-affected cattle blocked E-rosette augmentation induced by BOSCC extract, and autologous sera from three cattle with cutaneous papillomas blocked E-rosette augmentation induced by papilloma extract. Sera from cattle with BOSCC did not block E-rosette augmentation induced by papilloma extract, nor did sera from cattle with cutaneous papillomas block BOSCC extract-induced E-rosette augmentation. Sera from cattle with BOSCC blocked BOSCC extract-induced E-rosette augmentation in some but not all allogeneic combinations. E-rosette augmentation was observed when blood leukocytes from all 12 BOSCC-affected and six control cattle were exposed to phytohemagglutinin. This reaction was blocked by autologous sera from 11 of the 12 BOSCC-affected cattle, from one of the three cattle with cutaneous papillomas, but from none of the three other cattle. Sera from BOSCC-affected cattle also blocked phytohemagglutinin-induced E-rosette augmentation in allogeneic combination, regardless of the tumor status of the donor of the leukocytes.

The E-rosette augmentation (ERA) assay was used to study cell-mediated immunity with blood leukocytes from cattle with bovine ocular squamous cell carcinoma (BOSCC), from cattle with benign ocular or cutaneous lesions, and from healthy controls. Potassium chloride extracts (3 M) of BOSCC and skin from the same donor, cutaneous bovine papillomas, allogeneic lymphosarcoma, and xenogeneic sheep squamous cell carcinoma were used as antigens. Of the 21 animals with BOSCC, leukocytes of 19 gave positive ERA reactions to BOSCC extract and only those of 4 reacted nonspecifically to other extracts. Of 21 animals in the control group, only 3 gave positive ERA reactions. In reciprocal tests, cattle with BOSCC showed ERA reactivity only against extracts of BOSCC, and cattle with cutaneous papillomas showed reactivity only against extracts of cutaneous papillomas. Blood leukocytes from tumor-bearing cattle stimulated with related tumor extracts released a soluble factor that enhanced E-rosette formation when tested on normal bovine leukocytes.